SUMMARY (Project 1: Donna Zhang) Contamination of soil and water by metal-containing hazardous substances, particularly at sites near mine tailings and smelters, has led to chronic exposure of nearby communities to toxic metal mixtures, posing a serious health problem. Based on data from the Agency for Toxic Substances Disease Registry, the number one contaminant associated with mine tailings at these sites is the toxic metalloid arsenic (As). Epidemiological studies have demonstrated a positive correlation between chronic As exposure, either through drinking water or food, with an increased incidence of diabetes. Thus, exposure to As-containing mine tailings, which could result in inhalation or ingestion of As, may be a significant contributor to enhanced risk of disease in exposed communities. Importantly, despite the known severity of the health effects, the molecular mechanisms by which As-containing mine tailings enhance diabetic phenotypes have not yet been elucidated. Previously, we reported that low, environmentally relevant doses of arsenic block autophagy, a key cellular degradation pathway critical to maintaining proteostasis. Furthermore, we have shown that autophagic dysfunction results in prolonged activation of the key antioxidant transcription factor NRF2. Normally maintained at low levels through KEAP1-mediated ubiquitination and degradation by the 26S proteasome, NRF2 is upregulated at the protein level via oxidative modification of KEAP1 (KEAP1-C151 dependent, canonical) or sequestration of Keap1 into autophagosomes during As-induced autophagy dysfunction (p62-dependent, non-canonical). While controlled Nrf2 activation through the Keap1-C151 dependent canonical mechanism is protective, prolonged p62-dependent non-canonical activation of NRF2 during As exposure causes cellular dysfunction and tissue damage, indicative of a ?dark side? to NRF2. We hypothesize that As-containing mine tailings promote diabetes through p62-dependent, prolonged activation of Nrf2. This hypothesis is supported by our preliminary data indicating that wild type (WT) mice exposed to As showed impaired glucose tolerance and enhanced insulin resistance, which was not observed in Nrf2-/-, p62-/-, or Nrf2-/-p62-/- mice. Our recent RNAseq data generated from the liver of mice exposed to As for 20 weeks also showed significant changes in the expression of genes involved in glucose, insulin, cholesterol, and lipid metabolism. In this application, we will test our hypothesis by: 1) characterizing the time and dose-dependent diabetogenic potential of chronic exposure to As in drinking water or mine tailing As-particles (PM10) in WT mice (Aim 1); 2) determining the role of prolonged NRF2 activation in driving As-induced metabolic reprogramming in diabetes-relevant cell lines (Aim 2); and 3) in vivo confirmation of important molecular alterations induced by As and prolonged NRF2 activity in promoting diabetes (Aim 3). A mechanistic understanding of arsenic-mediated alterations that lead to diabetes will prove extremely valuable in the generation of diagnostic, preventive, and therapeutic strategies for populations exposed to As-containing mine tailings and populations at risk of arsenic exposure.